Microsomal enzymes are altered biochemically by nutritional deficiencies and excesses. Vitamin C deficiency in guinea pigs results in decreased activity of the liver microsomal monooxygenase system and the basis for this decrease will be investigated. The objectives of the proposed research are: 1) to determine the effects of ascorbic acid deficiency on the activity of NADPH-cytochrome P-450 reductase, a key component of the microsomal system and 2) to ascertain whether the reductase is the rate-limiting component in this system. Previous studies which measured reductase activity in crude microsomes were inconclusive because the decreases in activity seen in ascorbate-deficient microsomes may have resulted from decreased concentrations of the substrate, cytochrome P-450. In the present study the reductase activity will be separated away from its substrate and analyzed under conditions of substrate saturation. Liver microsomes will be prepared from guinea pigs maintained on an ascorbic acid-deficient diet for 21 days. Control guinea pigs will be fed the same diet but will receive ascorbic acid (1 mg/ml) in their drinking water. Possible qualitative differences in enzyme activity will be investigated by purifying the cytochrome P-450 reductases from control and ascorbic acid-deficient guinea pigs and characterizing the enzymes as to their kinetic properties, pH and ion requirements, molecular weights, and flavin compositions. Experiments incorporating purified reductase into microsomes will determine whether the enzyme is rate-limiting for monooxygenase reactions. In addition to elucidating effects of ascorbic acid deficiency, these studies should provide a basis for evaluating possible effects of excess ascorbate in mammalian systems.